||The neuromuscular junction (NMJ) is the synapse formed between a motor nerve and a muscle cell. A hallmark step in NMJ development is the postsynaptic clustering of muscle acetylcholine receptors (AChRs) that is orchestrated by nerve-secreted agrin and its receptor, muscle-specific kinase (MuSK). In the downstream signaling cascade initiated by MuSK, protein kinases feature prominently and bring about the phosphorylation, clustering and cytoskeletal anchoring of AChRs and associated proteins. In contrast, little is known about how protein phosphatases counter-balance the actions of MuSK and other protein kinases during NMJ development. For my thesis research I investigated the involvement of protein tyrosine and ser/thr phosphatases in regulating postsynaptic signaling at the NMJ. Using cultured C2 mouse myotubes I first showed that the general tyrosine phosphatase inhibitor Na-pervanadate functionally activated MuSK and enhanced both agrin-independent and agrin-dependent AChR clustering in muscle cells. Next, by immuno-screening I identified the SH2 domain-containing phosphatase Shp2 as a major tyrosine phosphatase in C2 myotubes and demonstrated that its selective down-regulation by RNA interference increased MuSK activation and AChR clustering. Additionally, I found that agrin-treatment of C2 myotubes increased the tyrosine phosphorylation and Shp2-interaction of the Shp2-activating protein SIRPα1, suggesting that Shp2 functions in a feedback loop to regulate agrin/MuSK signaling. Interestingly, depletion of Shp2 raised the basal tyrosine phosphorylation of SIRPα1 to levels normally obtained with agrin, indicating that SIRPα1 is also a substrate of Shp2 in muscle cells. Lastly, using pharmacological and molecular approaches I showed that the ser/thr phosphatase calcineurin positively regulates MuSK activation and agrin-induced AChR clustering in muscle cells. Taken together, my thesis research findings highlight the importance of protein phosphatases in postsynaptic signaling at the NMJ and provide insights into how balancing actions of protein kinases and phosphatases may determine the formation of synaptic specializations in the developing nervous system.